Biology subjects

Tanis, J. E.

Publications and source records attributed to Tanis, J. E..

8 recordsLinked to original sources

bioRxiv2025

Superoxide dismutase impacts extracellular vesicle biogenesis and uptake

Extracellular vesicles (EVs), which transfer bioactive macromolecules between cells, play an important role in the pathogenesis of multiple neurodegenerative diseases. Focus has centered on how altered EV contents propagate disease and the potential for EVs as diagnostic biomarkers, while the effect of pathogenic factors on EV release is less understood. Here, we defined how the key antioxidant enzyme superoxide dismutase 1 (SOD-1) affects EV shedding from sensory neuron primary cilia, enrichment of ciliary proteins packaged into EVs, and uptake of EVs by surrounding glia in vivo by imaging C. elegans expressing fluorescent protein-tagged EV cargos. We discovered that loss of SOD-1, as well as the SOD-1(G85R) amyotrophic lateral sclerosis (ALS) pathogenic variant, increased EV shedding from the cilium distal tip, and this was associated with greater abundance of EV cargo in this ciliary compartment. In contrast, loss of SOD-1 reduced the glial uptake of a different cargo present in EVs shed from the ciliary base. Together, this suggests that redox balance has a subtype-specific effect on EV biogenesis, influencing neuron communication in vivo.

cell biology↗

bioRxiv2025

C. elegans CED-1 acts in neurons to modulate ciliary protein abundance and extracellular vesicle shedding

The C. elegans receptor CED-1, along with orthologs mammalian MEGF10 and Drosophila Draper, plays a well-established and conserved role in phagocytosis by acting in engulfing cells.1-7 Interestingly, CED-1 family members are also expressed in neurons, but their functions in these non-engulfing cells remain unclear.6,8,9 Our study shows that CED-1 localizes to the dendrites and primary cilia of male tail RnB neurons, which mediate sensory perceptions during mating10 and generate extracellular vesicles (EVs) that transfer bioactive macromolecules for both intercellular and animal-to-animal communication.11 Loss of ced-1 leads to a reduction in the shedding of EVs that contain the transient receptor potential (TRP) channel PKD-2 from the cilium distal tip, and this is rescued by the expression of CED-1 in the neurons. CED-1 is required to increase both the abundance of PKD-2 in the cilium and PKD-2 EV shedding in response to the physiological stimulus of mating partners. Assessment of ced-1 mutant male mating indicates that neuronal CED-1 is also important for turning behavior, which helps the male tail to maintain contact with a mate. Collectively, these results reveal a new role for CED-1 in neurons as a regulator of EV biogenesis in response to environmental cues, optimizing the shedding of bioactive EVs for effective communication.

cell biology↗

bioRxiv2025

Phosphatidylinositol (4,5)-bisphosphate Impacts Ectosome Shedding from C. elegans Ciliated Sensory Neurons

Small secreted extracellular vesicles (EVs) mediate intercellular transport of bioactive macromolecules. How the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which plays a critical role in many cellular processes, impacts EV biogenesis is unclear. The primary cilium, a sensory organelle protruding from most non-dividing cells, transmits signals by shedding EVs called ectosomes. Here, we altered ciliary PI(4,5)P2 by manipulating expression of the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K1) PPK-1 and deletion of the phosphoinositide 5-phosphatase (INPP5E) inpp-1, then determined the impact on release of EVs that carried cargos tagged with fluorescent proteins. We discovered that increasing PI(4,5)P2 differentially affected ectosome shedding from distinct compartments, decreasing biogenesis of an EV subpopulation from the ciliary base, but enhancing budding from the cilium distal tip. Altering PI(4,5)P2 levels also impacted the abundance and distribution of EV cargos in the cilium, but not the sorting of the protein cargos into distinct subsets of ectosomes. Finally, manipulating PI(4,5)P2 did not affect cilium length, suggesting that changing PI(4,5)P2 levels can serve as a mechanism to regulate ectosome biogenesis in response to physiological stimuli without impacting cilium morphology. Summary StatementExtracellular vesicles (EVs), released from most, if not all, cell types, share bioactive cargo that cannot readily cross the plasma membrane with recipient cells. EVs play numerous roles in physiological processes as well as the propagation of pathophysiological conditions, including cancer, neurodegenerative, kidney, and cardiovascular diseases. Here, we sought to determine the impact of the membrane lipid phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) on EV biogenesis. We used a genetic approach to manipulate PI(4,5)P2 levels in the primary cilium, a specialized sensory organelle that serves as a platform for signal transmission by shedding EVs. We discovered that high PI(4,5)P2 differentially impacts two distinct EV subpopulations, decreasing shedding of ectosomes derived from the ciliary base, but increasing budding from the cilium distal tip. This work defines a new role for PI(4,5)P2 in the regulation of EV biogenesis and ciliary biology.

cell biology↗

bioRxiv2024

In-cell processing enables rapid and in-depth proteome analysis of low-input Caenorhabditis elegans

Caenorhabditis elegans is a widely used genetic model organism, however, the worm cuticle complicates extraction of intracellular proteins, a prerequisite for typical bottom-up proteomics. Conventional physical disruption procedures are not only time-consuming, but can also cause significant sample loss, making it difficult to perform proteomics with low-input samples. Here, for the first time, we present an on-filter in-cell (OFIC) processing approach, which can digest C. elegans proteins directly in the cells of the organism after methanol fixation. With OFIC processing and single-shot LCMS analysis, we identified over 9,400 proteins from a sample of only 200 worms, the largest C. elegans proteome reported to date that did not require fractionation or enrichment. We systematically evaluated the performance of the OFIC approach by comparing it with conventional lysis-based methods. Our data suggest equivalent and unbiased performance of OFIC processing for C. elegans proteome identification and quantitation. We further evaluated the OFIC approach with even lower input samples, then used this method to determine how the proteome is impacted by loss of superoxide dismutase sod-1, the ortholog of human SOD-1, a gene associated with amyotrophic lateral sclerosis (ALS). Analysis of 8,800 proteins from only 50 worms as the initial input showed that loss of sod-1 affects the abundance of proteins required for stress response, ribosome biogenesis, and metabolism. In conclusion, our streamlined OFIC approach, which can be broadly applied to other systems, minimizes sample loss while offering the simplest workflow reported to date for C. elegans proteomics analysis.

biochemistry↗

bioRxiv2021

Kinesin-II motors differentially impact biogenesis of distinct extracellular vesicle subpopulations shed from C. elegans sensory cilia

Extracellular vesicles (EVs) are bioactive lipid-bilayer enclosed particles released from nearly all cells. One specialized site for EV shedding is the primary cilium, a conserved signaling organelle. The mechanisms underlying cargo enrichment and biogenesis of heterogeneous EVs shed from cilia are unclear. Here we discover the conserved ion channel CLHM-1 as a new ciliary EV cargo. Using super-resolution microscopy, we imaged EVs released into the environment from sensory neuron cilia of C. elegans expressing fluorescently-tagged CLHM-1 and TRP polycystin-2 channel PKD-2 EV cargoes at endogenous levels. We find that these proteins are enriched in distinct EV subpopulations that are differentially shed in response to availability of hermaphrodite mating partners. Both CLHM-1 and PKD-2 localize to the ciliary base and middle segment of the cilium proper, but PKD-2 alone is present in the cilium distal tip and EVs shed from this site. CLHM-1 EVs released into the environment bud from a secondary site, the periciliary membrane compartment at the ciliary base. We show that individual heterotrimeric and homomeric kinesin-II motors have discrete impacts on the colocalization of PKD-2 and CLHM-1 in both cilia and EVs. Total loss of kinesin-II activity significantly decreases shedding of PKD-2 but not CLHM-1 EVs. Our data demonstrate that anterograde kinesin-II-dependent intraflagellar transport is required for selective enrichment of specific protein cargoes into heterogeneous EVs with different signaling potentials.

cell biology↗

bioRxiv2021

Dietary vitamin B12 reduces amyloid-β proteotoxicity by alleviating oxidative stress and mitochondrial dysfunction

Alzheimers disease (AD) is a devastating neurodegenerative disorder with no effective treatment. Diet, as a modifiable risk factor for AD, could potentially be targeted to slow disease onset and progression. However, complexity of the human diet and indirect effects of the microbiome make it challenging to identify protective nutrients. Multiple factors contribute to AD pathogenesis including amyloid beta (A{beta}) deposition, mitochondrial dysfunction, and oxidative stress. Here we used Caenorhabditis elegans to define the impact of diet on A{beta} proteotoxicity. We discovered that dietary vitamin B12 alleviated mitochondrial fragmentation, bioenergetic defects, and oxidative stress, delaying A{beta}-induced paralysis without affecting A{beta} accumulation. Vitamin B12 had this protective effect by acting as a cofactor for methionine synthase rather than as an antioxidant. Vitamin supplementation of B12 deficient adult A{beta} animals was beneficial, demonstrating potential for vitamin B12 as a therapy to target pathogenic features of AD triggered by both aging and proteotoxic stress.

cell biology↗

bioRxiv2020

A C. elegans genome-wide RNAi screen for altered levamisole sensitivity identifies genes required for muscle function

At the neuromuscular junction (NMJ), postsynaptic ionotropic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. To identify regulators of postsynaptic function, we conducted a genome-wide RNAi screen for genes required for proper response to levamisole, a pharmacological agonist of ionotropic L-AChRs at the Caenorhabditis elegans NMJ. A total of 117 gene knockdowns were found to cause levamisole hypersensitivity, while 18 resulted in levamisole resistance. Our screen identified conserved genes important for muscle function including some that are mutated in congenital myasthenic syndrome, congenital muscular dystrophy, congenital myopathy, myotonic dystrophy, and mitochondrial myopathy. Of the genes found in the screen, we further investigated those predicted to play a role in endocytosis of cell surface receptors. Loss of the Epsin homolog epn-1 had opposing effects on the levels of postsynaptic L-AChRs and GABAA receptors, resulting in increased and decreased abundance, respectively. This disrupts the balance of postsynaptic excitatory and inhibitory signaling, causing levamisole hypersensitivity. We also examined other genes that resulted in a levamisole hypersensitive phenotype when knocked down including gas-1, which functions in Complex I of the mitochondrial electron transport chain. Consistent with altered ATP synthesis impacting levamisole response, treatment of wild-type animals with levamisole resulted in L-AChR dependent depletion of ATP levels. These results suggest that the paralytic effects of levamisole ultimately lead to metabolic exhaustion.

genetics↗

bioRxiv2020

A rapid, super-selective method for detection of single nucleotide variants in C. elegans

With the widespread use of single nucleotide variants generated through mutagenesis screens, the million mutation project, and genome editing technologies, there is pressing need for an efficient and low-cost strategy to genotype single nucleotide substitutions. We have developed a rapid and inexpensive method for detection of point mutants through optimization of SuperSelective (SS) primers for end point PCR in Caenorhabditis elegans. Each SS primer consists of a 5 "anchor" that hybridizes to the template, followed by a non-complementary "bridge," and a "foot" corresponding to the target allele. The foot sequence is short, such that a single mismatch at the terminal 3 nucleotide destabilizes primer binding and prevents extension, enabling discrimination of different alleles. We explored how length, stability, and sequence composition of each SS primer segment affected selectivity and efficiency in order to develop simple rules for primer design that allow for distinction between any mismatches in various genetic contexts over a broad range of annealing temperatures. Manipulating bridge length affects amplification efficiency, while modifying the foot sequence can increase discriminatory power. Flexibility in the positioning of the anchor enables SS primers to be used for genotyping in regions with sequences that are challenging for standard primer design. In summary, we have demonstrated flexibility in design of SS primers and their utility for genotyping in C. elegans. Since SS primers reliably detect single nucleotide variants, we propose that this method could have broad application for SNP mapping, screening of CRISPR mutants, and colony PCR to identify successful site-directed mutagenesis constructs.

genetics↗